JPH0218070B2 - - Google Patents

Abstract

Disclosed is a method for the immobilization of an enzyme producing microorganism or an enzyme produced thereby. The method involves fiocculating the biocatalyst with polyethylenimine and adding chitosan and glutaraldehyde to form a reaction product. In a preferred embodiment, the reaction product is extruded onto the revolving plate of a spheronizing device to form spherical enzyme aggregates having enhanced physical and biocatalytic properties.

Description

[Detailed description of the invention]

The use of enzymes derived from microbial cells to effect specific chemical transformations is known. In carrying out such conversions, biocatalysts [biocatalysts;
Cell-free enzyme preparations, disrupted cells or whole cells can be effectively used as raw materials for biocatalysts. Although free enzymes or cells can be used effectively in batch processes, they are not useful for continuous industrial scale operations. for example,
In the conversion of glucose to fructose catalyzed by glucose isomerase or the conversion of sucrose to palatinose catalyzed by sugar mutase, an economically competitive process involves converting the substrate solution (glucose or sucrose) onto a fixed bed of biocatalysts. and continuous recovery of the converted product through a fixed bed. This process technology has stimulated interest in the preparation of various forms of immobilized biocatalysts. For example, the use of polyethyleneimine and glutaraldehyde for the immobilization of glutaraldehyde-sensitive enzymes
Disclosed by Gestrelius in US Pat. No. 4,288,552. An improvement to this method was published in a U.S. patent.
No. 4,355,106, in which an intracellular enzyme that is not sensitive to glutaraldehyde is first brought into contact with glutaraldehyde and then immobilized by introducing polyethyleneimine into the reaction medium. In U.S. Patent No. 4,167,447, an aqueous solution of an enzyme was dissolved in chitosan and PH3.
A method for immobilizing an enzyme is disclosed that includes mixing at 7 to 7 to produce a product that can be precipitated by addition of an alkali or sulfate ion feedstock.
In another embodiment of the method disclosed in this patent, solid chitosan is cross-linked with a multifunctional cross-linking agent such as a dialdehyde and then contacted with an aqueous solution of an enzyme. Enzyme by Muzzarelli
Microb. Technol., 1980, Volume 2, July 1977 discloses the immobilization of enzymes including glucose isomerase onto chitin and chitosan. JP-A No. 51 [1976]-128, 474 discloses a method for immobilizing glucose isomerase, which involves blending glucose isomerase-producing bacteria and chitosan, followed by treatment with polyaldehyde. No. 4,094,743 describes the preparation of enzymatically active products insoluble in aqueous media by treating chitosan as an inert support with dialdehyde and subsequently immobilizing the enzyme on the thus treated support. The method is described. Related patent, U.S. Patent Application No. 467,851 (1983
Immobilization of the biocatalyst using a polyamine flocculating agent and a cross-linking agent (dated February 24, 2013), the cylinder was placed in a cylinder such that it could rotate freely with the cylinder forming a stationary side wall relative to the plate. The subsequent spheronization of the immobilized product using a spheronizing device consisting of a notched friction plate as a rotor is disclosed. German Patent Application No. 21 37 042 describes a method for spheronizing enzyme compositions, especially those useful in the detergent industry. The equipment used for such spheronization is similar to that described in the above-mentioned US Pat. No. 4,678,51. No. 4,283,496, Flavobacterium arborescens
A method for converting glucose to fructose is disclosed that involves the use of enzymes produced by microorganisms from the species S. arborescens. The present invention provides an aqueous medium containing a glucose isomerase or a population of bacterial cells containing intracellular glucose isomerase having a molecular weight of at least about 500 daltons and at least 10 of the amino groups of the polymer.
introducing an aqueous solution of polyethyleneimine (PEI) having a primary amino group content of % by weight; b. adding an aqueous solution of glutaraldehyde and chitosan to the aqueous medium to form a crosslinked reaction product; and c. an enzyme-producing microorganism, or produced from an enzyme-producing microorganism, comprising forming a reaction product by removing the bound reaction product from the aqueous medium and drying the same to provide the desired product having biocatalytic activity. This is a method for immobilizing enzymes. The method of the invention can be applied to, for example, B. licheniformis, S. olivaceous.
and A. missouriensis. For example, when F. arborescens cells are homogenized to solubilize 40% of their glucose isomerase activity, complete immobilization of the soluble enzyme is observed. Cells to be fixed are typically grown in an aqueous nutrient medium and concentrated by either filtration or centrifugation to provide an aqueous medium containing about 10% (by weight) dry matter. Cells containing enzymes or extracellular enzymes in the case of disrupted cells are condensed by adding polyethyleneimine, chitosan and glutaraldehyde to an aqueous medium. The polyethyleneimine used should have a molecular weight of at least about 500 Daltons, with a maximum molecular weight of 100,000 Daltons preferred. A preferred molecular weight range is about 40,000 to 60,000 Daltons. However, immobilization performed using low molecular weights (approximately 40,000 daltons or less) resulted in nodule aggregation, but the aggregated cell populations could not be easily recovered by filtration. It was expected that the actual chemistry of immobilization would be similar to that performed with high molecular weight PEI. The physical properties of cell aggregates are such that cell aggregates clog filter paper (bind).
off), so they look different to some extent. Another common recovery method is centrifugation. Since the aggregated cell aggregates were virtually insoluble, they could be recovered by centrifugation. The filled cell aggregates were then extruded and molded in a conventional manner to obtain the desired immobilized enzyme particles. The primary amino groups of the polymer should be at least about 10% by weight of the total amino group content of the polymer, with a preferred amount being about 25%. It is customary for the polyethyleneimine to be present as an aqueous solution, preferably at a concentration of 1 to 10% by weight, before being added to the enzyme-containing medium. In this way, proper mixing of PEI in the cell mixture is achieved. Typically, the amount of polyethyleneimine added will be 2-22% by weight of the enzyme preparation to be immobilized. A cross-linked reaction product is generated by adding an aqueous solution of glutaraldehyde and chitosan to this medium. These reactants can be added in any order or simultaneously. However, if the highest hardness of immobilized enzyme particles is desired, glutaraldehyde should be added first. In some cases, glutaraldehyde can partially deactivate the enzyme, so chitosan is added first if maximum activity is desired. As used herein, the term chitosan refers to a polyaminopolysaccharide obtained by N-deacetylation of chitin using strong alkali and heat. Chitin is a polysaccharide whose primary repeating unit in the molecule is 2-deoxy-2-(acetylamino)-glucose. Generally, about one unit out of every six polymerized units of chitin is unacetylated, whereas in chitosan essentially all repeating units are unacetylated. The degree of unacetylation can be controlled by the intensity of the deacetylation reaction. Chitin, which is widely distributed in nature such as marine invertebrates, insects, molds, and yeasts, is easily prepared by removing impurities from the shells of crabs, shrimp, lobsters, crayfish, etc., which are obtained in large quantities from seafood processing plants. Ru. Typically, the amount of chitosan used is 0.5-22% by weight of the immobilized enzyme preparation, with amounts of 3.5-10% being preferred. Before adding chitosan to the enzyme containing medium it must be placed in an aqueous solution since it is usually poorly soluble in water. However, chitosan is soluble in dilute aqueous acids; ie, inorganic acids with the exception of formic acid, acetic acid, pyruvic acid, lactic acid, maleic acid, citric acid, and sulfuric acid, which yields insoluble chitosan sulfate complexes. For the immobilization method of the invention to be effective, chitosan must become a constituent part of the immobilized cell complex. Therefore, chitosan is placed in an aqueous solution state before use. about
Solutions containing 0.2-1.0% by weight chitosan are preferred and are prepared by adding chitosan to a 0.5% solution of acetic acid. Heating this solution to about 60°C promotes dissolution of chitosan. After heating, the chitosan solution is filtered to remove insoluble residues. The glutaraldehyde used in this immobilization method is used to prevent local concentrations that can inhibit enzymes during reagent addition. Glutaraldehyde is used in an amount of 4-26% by weight of the immobilized enzyme;
Amounts of 7 to 15% by weight are preferred. The biocatalyst, polyethyleneimine, chitosan, and glutaraldehyde are combined to form a bioactive reaction product that can be removed from the reaction medium by liquid/solid separation techniques, which can be dried and crushed into particles. , and used for biocatalytic conversion. However, this solid reaction product was dried to yield a wet cake containing approximately 68-76% water by weight, and this wet cake was placed in a cylinder such that the cylinder constituted a stationary wall for the plate. It has been found that a more desirable physical shape of the material can be obtained by having a notched friction plate as a rotor and extruding it onto a rotating plate of a spheronizer, which plate is free to rotate. The filter cake is extruded onto a rotating notched plate through an orifice with a diameter size approximately equal to that desired for the spherical particles, where it is shaped like an annular or toroidal plate with a cross-sectional area sloped to the cylinder wall. arranged in a shape. The extrudate is initially broken into short pieces, ideally of comparable diameter, by frictional forces on the notched plate and also by interparticle collisions and friction of the moving mass. The characteristic configuration of this material results from the centrifugal force due to the rotation of the plate and the tangential force of the friction between the material and the scored surface. The smooth rolling surface cannot roll up the extrudate, but only slides it around the plate. The characteristics of this movement are different, so when using a smooth plate, the final shape of the material is not as uniform and spherical as desired. Spheronizers of the type described above are sphere-making machines that can rapidly convert immobilized enzyme extrudates into small, dense, easily handled spheres. The extrudate should preferably have a moisture content within the above range. A moisture content that is too low will cause particle crushing during the spheronization process, causing the formation of particles that are too small. Too much moisture causes fouling and coagulation problems during the spheronization process, leading to the formation of particles that are too large. The spheronizer used in the following examples is a Marumerizer Q.
−230 under the trademark Fuji Padal Co., Ltd., Osaka;
It is commercially available from Japan. This device has a rotor with a diameter of 23 cm. The preferred tangential speed for producing spheres from extrudates used in the present invention is 4.5-12
m/sec (Tangential velocity = RPM ÷ 60 x πX; X is the diameter of the plate). Therefore, to achieve the desired spheronization result, the rotation speed of the 23 cm diameter plate is between 500 and 1000 RPM. If the rotational speed is too high, the plate will tend to slide the extruded enzyme mass towards the cylinder wall;
On the other hand, if the rotation of the plate is too small, it will not provide enough momentum in the rotating mass for collisions and friction as forces to refine and spheroidize the desired spheroidization. not achieved. After spheronization, the cell aggregates are typically dried in a fluid bed dryer. The enzyme activity of the spheroidized enzyme aggregates is approximately equal to or greater than that of the ground particles;
This shows that this spheronization method is advantageous in providing particles with suitable biocatalytic activity. Apart from providing spherical particles that are inherently easier to handle than their crushed forms, the spheres prepared by this method exhibit greater physical robustness and long-term enzyme productivity in the reactor. The method of carrying out the invention is further illustrated in the following examples: Example 1 Liquid consisting of 7% corn steep, 1.4% sweet whey powder and 0.15% yeast extract Adjust the medium to PH8.3,
1000 ml of the resulting product was put into a fermenter containing 1500 ml. This medium was sterilized at 121°C for 60 min, inoculated with F. arborescens strain ATCC4358, and then incubated at 30°C.
The cells were cultured for 18 hours. The cells containing glucose isomerase are then placed in a self-cleaning bowl.
The fermented broth was concentrated by centrifugation on a Westfala Model SAMR5036 centrifuge using This concentrate was estimated to contain approximately 10% dry matter. Virtually no cell disruption occurred during centrifugation. The PH value of this cell slurry
Adjust to 8.0, then heat to 70℃, and at 70℃
It was held for 15 minutes and then cooled to about 25°C. Glucose isomerase activity was hardly lost during this heat treatment. This heat treatment is performed in the following two ways.
It served two important functions: 1) to facilitate recovery of cellular material after fixation, and 2) to inactivate proteases entrained and produced by cells. Add 1000 ml of deionized (DI) water to 200 ml of heat-treated cell slurry with stirring, then add 5% (w/v) PEI (P
-600Cardova Chemical Co.) 40ml was added.
After thoroughly mixing PEI into slurry, 10%
20 ml (w/v) glutaraldehyde (prepared from 25% commercially available glutaraldehyde) was thoroughly mixed into this slurry. Next, 200 ml of 0.25% (w/v) chitosan solution was added. PH value after chitosan addition is 7.8
This was adjusted to 8.0 with diluted sodium hydroxide. The PH value during this immobilization step ranged from 7.0 to 9.0. 8.0 for this particular microorganism.
The PH value is mainly selected, and the reason is that below PH7.5, the PH value
This is because the setting process is not accelerated as in 8.0. The chitosan solution was prepared by mixing the appropriate amount of chitosan in DI water to a 1% solution. Glacial acetic acid was added to a final concentration of 0.5% and the mixture was then heated to 60° C. with thorough mixing. Virtually all of the chitosan dissolved during this heating step. Next, add this hot solution to
Dilute 4x with DI water and then add on a Buchner funnel.
Filtered through Shirkskin filter paper. The filtrate is heated to about 25℃
and then the PH value was carefully adjusted to 5.0 with IN sodium hydroxide. After chitosan addition, the slurry was kept stationary for 1 hour. at this point
The cell population was collected on a Buchner funnel and the cell cake was extruded through a die with a 1.0 mm opening. 60
After further dehydration of the extruded material by drying at Measured for toughness. Glucose isomerase activity in these particles was determined by placing 0.1 g of the enzyme in a 250 ml Erlenmeyer flask, and adding a substrate [2M
Glucose, 20mM MgSO ₄ 7H ₂ O, 0.5mM
50 _ml of _CoCl2.6H2O and 20mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer was added. The dried particles were allowed to hydrate for approximately 1 hour at room temperature. At zero time,
The flask was placed in a 60°C water bath. The flask was agitated using a shaking water bath at a rate sufficient to move the particles. At 10 and 70 minutes, samples of 0.1 ml were removed from the measurement mixture and 4.0 ml of 0.1 MHClO ₄ were added. The amount of fructose produced was measured using the cysteine-H ₂ SO ₄ method (Dische, ZJBiological Chemistry, Vol. 181,
379, 1949) by incubating 0.05 ml of the sample with 0.5 ml of 5% cysteine and 4.5 ml of 75% H ₂ SO ₄ at 37° C. for 30 minutes and reading the absorbance at 412 nm. An immobilized glucose isomerase unit (IGIU) is the amount of enzyme that produces 1 micromole of fructose per minute. Particle toughness is determined using the Instron Universal Tester
It was determined by measuring the work required to compress 20 to 40 mesh particles hydrated in 2M glucose at pH 8.0 using Model 1102. Details of this method can be found in related patent application No. 467,851.
No. The toughness of the particles is given in Kg-in. When 200ml of cell slurry was treated with the above fixation method, the activity was 421 IGIU per 1g and 3.9Kg-
This yielded 21.1 g of dry material with a particle toughness of in. Examples 2-5 Different amounts of PEI were added to the heat-treated cell slurry of Example 1 before adding glutaraldehyde and chitosan. After adding the reagents for immobilization, the cell population was collected, extruded, and dried as in Example 1. Table 1 below shows particle integrity and activity at various PEI concentrations.
It is shown that particle integrity and immobilized enzyme yield improve as the amount of PEI increases.

TABLE *Particle toughness as % of particle toughness of Example 1 Examples 6-24 After addition of PEI as described in Example 1, different amounts of glutaraldehyde were added to the cell slurry. 2
The effect of seed amount on PEI activity and particle integrity is shown in Table 2. It can be seen that the particle toughness increases as the amount of glutaraldehyde increases. It is found that the activity yield is inversely proportional to the amount of glutaraldehyde. The apparent loss of activity may be caused by the chemical reaction of glutaraldehyde with the enzyme taking place to deactivate the enzyme, or by the fact that glutaraldehyde can increase the mass transfer resistance of substrates and products of the enzymatic reaction. was completed. The results in Table 2 also show that even larger populations of immobilized enzyme can be obtained at higher amounts of PEI.

[Table] Examples 25 to 30 Immobilization was performed by changing the amount of chitosan. The method described in Example 1 was used using two different amounts of PEI and glutaraldehyde. The effect on activity and particle toughness is shown in Table 3. The two PEI levels improved activity and particle toughness as the amount of chitosan increased. At high amounts of glutaraldehyde, particle toughness was improved, while activity was not improved at high amounts of chitosan. In the immobilization step (Examples 28-30), the third PEI
Various amounts of chitosan were used. The results shown in Table 3 show that the enzyme yield decreases when the amount of chitosan exceeds the optimum amount.

[Table] Examples 31 to 35 Using the immobilization method described in Example 1 with various molecular weights.
A PEI sample was used. Results are reported in Table 4 showing that high molecular weight PEI works best. Immobilization using low molecular weight PEI resulted in a type of aggregated cell population that could not be recovered by conventional filtration methods. Appropriate aggregates could be obtained by varying the amounts of PEI and/or glutaraldehyde and/or chitosan. 12, 18, 150 and 600 in the molecular weight column in Table 4
indicates that it has an average molecular weight that is 10 ² times these values.

[Table] **See Table 1 Examples 36 to 40 The order of adding glutaraldehyde and chitosan in Example 1 was changed. Table 5 shows the activity and particle toughness observed by interchanging the order of addition of glutaraldehyde and chitosan at various amounts of PEI. Greater immobilized enzyme activity was obtained when glutaraldehyde was added last, but the particle toughness was reduced to some extent.

【table】

[Table] *See Table 1 Example 41 The heat-treated cell slurry prepared as described in Example 1 was diluted with twice the volume of water. Add 15g of undiluted cell slurry to this cell slurry.
PEI, 2.5 g chitosan and 7.5 g glutaraldehyde were added using the method of Example 1. The cell aggregates were collected and dehydrated to approximately 75% moisture in a 12 inch basket centrifuge. Although the fermentations were carried out under conditions as identical as possible, the cells isolated from the various fermentations are not completely identical as far as the organization of the immobilized population is concerned. Therefore, depending on the batch, the suitable moisture content to produce spherical particles varies somewhat between 71 and 75% moisture. The cell cake was placed on a rotating plate of a spheronizer that could rotate freely, consisting of a notched friction plate as a rotor installed in the cylinder so that the cylinder provided a stationary wall against the plate. Extruded through a die. This notched plate has a diameter of 23 cm and a configuration of 2 mm pitch and 1 mm height.
Milled with. 7.2 per second of this plate
It was rotated at a speed of 600 RPM to give a tangential speed of m, after which the spheroidized extrudate was removed from the machine through a hole by centrifugal force. The spheronized particles were then dried in a fluid bed dryer (Aeromatic AG, Model No. 1) at an inlet air temperature of approximately 68°C. This method produced 108 g of dry material per cell slurry. This immobilized glucose isomerase preparation was measured at 437 IGIU/g of 20/40 mesh particle size;
It had a particle toughness of 442 (compared with Example 1 as 100). 10g portion of this immobilized enzyme
(30/40 mesh) with 4.1mM magnesium and
40% (w/w) DS (dry solids) containing _250ppmSO2
The mixture was hydrated for 1 hour at pH 8.0 and 25°C. The hydrated oxygen slurry was then placed in a 60°C water bath for approximately 2 hours and then transferred to a 100 x 1.5 cm glass jacketed column. Before adding the enzyme particles to the column, a small stopper made of glass wool was placed on the bottom plate and then 5 ml of 20/40 mesh alumina particles were added. The column temperature was maintained at 60°C. Glucose feed was then perculated down through this bed at a flow rate that maintained fructose conversion at 42-44%. Table 6 shows the activity over time and the productivity in g of DS of 42% fructose per g of enzyme. This decreasing trend of activity takes about 30 minutes to reach the half-time.
It shows that 58 days are required. At this half-life, approximately 5060 g of DS (42% fructose) was produced per gram of immobilized enzyme.

【table】

[Table] Example 42 This example shows that extracellular enzymes can be immobilized using this immobilization method. Cell juice of F. arborescens (NRRL B11022) was centrifuged at room temperature. This cell slurry
The culture broth was diluted to 34% by volume with DI water. This diluted cell slurry was homogenized in a Manton-Gaulin homogenizer at 8000-10000 psi. The cell material was passed through a homogenizer three times. The degree of enzyme solubilization by homogenization is shown in the following table. Table 7 Substances % of total soluble activity Cell slurry 2 1st homogenate 31 2nd homogenate 52 3rd homogenate 58 Fixed filtrate 4 The cell material homogenized three times was then homogenized as follows: Fixed to. The homogenized cell slurry was adjusted to PH8.0, then incubated at 70°C for 15 minutes, and then incubated at approximately 25°C.
It was cooled to 5% PEI (P-600, Cordova) 1038
ml, followed by 3460 ml of 0.25% chitosan, and finally 347 ml of 10% glutaraldehyde for cross-linking. Then the PH value of the cell mixture was adjusted to 8.0. Upon addition of glutaraldehyde, complete cell aggregation occurred, breaking from the clear supernatant. This aggregated cellular material was collected in a 12% basket centrifuge. The cell cake was then extruded and shaped into spherical particles by the method described in Example 41. It was found that the glucose isomerase activity remaining in the filtrate was only 4% of the total activity (7th
table). This result indicates that virtually all soluble activity was immobilized. The results of immobilization are shown in the following table.

[Table] *See Table 1 Example 43 Cultured broth of Streptomyces olivaceas containing glucose isomerase was immobilized as follows: Cultured broth from S. olivaceas fermentation was used for immobilization. The growth bodies of S. olivaceas mainly consist of mycelium, while in the case of F. arborescens the growth bodies are in the form of cells. Adjust cultured meat juice 2 to PH9.0,
Subsequently, 20 ml of 10% PEI (P-600, Cordova) was added, which caused a small amount of coagulation. Then 10%
Addition of 30 ml of glutaraldehyde did not result in an obvious increase in coagulation. Next, 0.25% chitosan
1.5 was added, which resulted in complete mycelial aggregation. The immobilized cellular material was collected by filtration and extruded as in Example 1. The table below summarizes the results.

[Table] *See Table 1 Example 45 Immobilization of Bacillus licheniformis mutant glucose isomerase. The next one is chitosan-glutaraldehyde-
An immobilization method is shown in which reagents are added in the order of PEI. B.
The growth of licheniformis is similar to that of F. arborescens; ie cellular and not mycelial. PH
20 ml of 10% glutaraldehyde was added to cultured meat juice 1 adjusted to 7.0. The cell mixture was diluted twice with water and then 25 ml of 5% PEI (Kodak, molecular weight range not specified) was added, which caused aggregation of the cells. Collect the fixed cell population by filtration,
It was then further processed as in Example 1. The table below summarizes this immobilization method.

[Table] Example 45 B.licheniformis (B.licheniformis,
Immobilization of glucose isomerase from ATCC31667). This example shows an immobilization method in which the reagents are added in the order GA-chitosan-PEI. 1% glutaraldehyde in 1 cultured meat juice adjusted to PH7.5
250 ml was added during which time the PH value was maintained at 7.5. Next, 0.2% chitosan was added to the cultured broth, followed by 5% chitosan.
38.4 ml of PEI (Kodak, molecular weight range unknown) was added.
Cell aggregates were collected by filtration and further processed as in Example 1. The table below summarizes this immobilization method.

[Table] Example 46 Another example of immobilization of glucose isomerase produced from B. licheniformis ATCC31667.
For this example, B. licheniformis culture broth 4 was adjusted to pH 7.5 and then incubated at 70°C for 15 minutes. After cooling to about 25°C, 0.2% chitosan 2 was then added. The PH value of the cell mixture was then adjusted to 9.0 before adding 100 ml of 10% glutaraldehyde.
Next, 80 ml of 5% PEI was added. The PH value of the mixture was maintained at 9.0 during the addition of glutaraldehyde and PEI. The aggregated cell population was collected by filtration, extruded and dried as in Example 1. The results of this immobilization method are shown in the following table.

[Table] **See Table 1

Claims (1)

[Scope of Claims] 1 a. In an aqueous medium containing glucose isomerase or a bacterial cell containing glucose isomerase in the cell, the polymer has a molecular weight of at least 500 daltons and at least one of the amino groups of the polymer
introducing an aqueous solution of polyethyleneimine (PEI) having a primary amino group content of 10% by weight; b adding an aqueous solution of glutaraldehyde and chitosan to the aqueous medium to form a cross-linked reaction product; and c A glucose isomerase-producing microorganism or the like, comprising forming a reaction product by removing the cross-linked reaction product from the aqueous medium and drying it to give a desired product having biocatalytic activity. A method for immobilizing glucose isomerase produced by. 2. The method according to claim 1, wherein the PEI has a molecular weight range of 40,000 to 60,000 Daltons. 3 1-10% by weight before introducing PEI into the aqueous medium
Claim 1 is placed in an aqueous solution state at a concentration of
The method described in section. 4. The method of claim 1, wherein the amount of PEI is between 2 and 22% by weight of the cell population to be immobilized. 5. Claim 1, wherein the amount of chitosan used is between 0.5 and 22% by weight of the enzyme preparation to be immobilized.
The method described in section. 6. The method according to claim 5, wherein the amount of chitosan is 3.5 to 10%. 7. The method of claim 1, wherein the amount of glutaraldehyde used is between 4 and 26% by weight of the immobilized enzyme preparation. 8 The amount of glutaraldehyde is 7 to 15%,
The method according to claim 7. 9 Glucose isomerase-producing microorganisms are Flavobacterium and F. arborescens.
The method according to claim 1, wherein the method is of a bacterial strain. 10. The method of claim 9, wherein said strain is described as ATCC4358. 11a in an aqueous medium containing glucose isomerase or bacterial cells containing glucose isomerase in the cell, having a molecular weight of at least about 500 daltons and having a primary amino group content of at least 10% by weight of the amino groups of the polymer. introducing an aqueous solution of polyethyleneimine (PEI); b providing glutaraldehyde in an amount of 4-26% of the immobilized enzyme preparation and chitosan in an amount of 0.5-22% by weight of the immobilized enzyme preparation; adding a sufficient amount of an aqueous solution of chitosan to form a cross-linked reaction product; c. removing the cross-linked reaction product from the aqueous medium and partially drying it to form a wet cake; and d feeding the wet cake through an orifice onto the plate of a spheronizing device consisting of a cylinder constituting a stationary wall and a knurled friction plate as a rotor located within the cylinder, while rotating the plate. , thereby forming a desired product. 12. The method of claim 11, wherein the wet cake contains 68-76% water by weight. 13. The method of claim 11, wherein the plate rotates at a speed sufficient to provide a tangential velocity of 4.5 to 12 meters per second.